Phosphorylation of serine, threonine and tyrosine residues by protein kinases represents one of the most common post-translational regulatory modifications of proteins. More than 200 protein kinases have been described, following either purification to homogeneity or molecular cloning (see, Hunter, T. (1991), Methods Enzymol., 200:3-37; Hanks, S. K., et al. (1991), Methods Enzymol., 200:38-81; Hanks, S. K. 1991), Curr. Opin. Struct. Biol., 1:369-383; and Hubbard, M. J., et al. (1993) Trends Biochem. Sci., 18:172-177). It is thought that as much as 2-3% of eukaryotic genes encode protein kinases. The importance of protein kinases in physiological processes has stimulated an active search for specific inhibitors with potential pharmnacological interest (see, Hidaka, H., et al. (1992), Annu. Rev. Pharmacol. Toxicol., 32:377-397). Several classes of compounds have been identified, such as staurosporine, naphthalene sulfonamides (W 7, ML-9, SC-9), isoquinoline derivatives (H-7, H-8, KN-62), sphingosine, tyrphostins and others, but in most cases these inhibitors display broad specificity. Only some pseudosubstrate autoinhibitory peptides show a high degree of specificity.
Cyclin-dependent kinases (CDK), in particular, have recently raised considerable interest in view of their essential role in the regulation of the cell division cycle (CDC) (see, Nigg, E. A. (1993), Trends in Cell Biol., 3:296-301; and Sherr, C. S. (1993), Cell, 73:1059-1065). CDKs are highly conserved among eukaryotic species. Higher eukaryotic cells contain several isoforms of CDKs that become activated in specific phases of the cell cycle. CDKs consist of a catalytic subunit, the prototype of which is CDC2, and a regulatory subunit (cyclin). Six human CDK proteins have been described so far (see, Meyerson, M., et al. (1992), EMBO J., 11:2909-2917; Meyerson, M., et al. (1994), Mol. Cell. Biol., 14:2077-2086; and Van den Heuvel, S., et al. (1993), Science, 262:2050-2054), namely, CDK1 (also known as CDC2) and CDK2-6. With the exception of CDK3, for which the regulatory cyclin has not yet been identified, all these CDKs proteins are regulated by the transient association with one member of the cyclin family, i.e., cyclin A (CDC2, CDK2), B1-B3 (CDC2), D1-D3 (CDK2, CDK4, CDK5, CDK6), E (CDK2). Each step of the cell cycle is thought to be regulated by such CDK complexes: G1/S transition (CDK2/cyclin E, CDK3/unknown cyclin, CDK4/cyclin D1-D3, CDK6/cyclin D3), S phase (CDK2/cyclin A), G2 (CDC2/cyclin A), G2/M transition (CDC2/cyclins B).
CDKs are able to phosphorylate many proteins involved in cell cycle events, including histones, lamins and tumor suppressor proteins, such as the retinoblastoma gene product pRb (see, Norbury, C., et al., supra, Matsushime, H., et al. (1992), Cell, 71:323-334, Nigg, E. E. (1993), Curr. Opin. Cell. Biol., 5:187-193). In accordance with their central role in the cell cycle, enzyme activity is tightly controlled by multiple mechanisms. Kinase activation requires complex formation with regulatory cyclin proteins as described above, followed by an activating phosphorylation on Thr-161 in CDC2 or the corresponding Thr in the other CDKs (see, e.g., Gould, K. L., et al. (1991), EMBO J., 10:3297-3309; Desai, D., et al. (1992), Mol. Biol. Cell, 3:571-582; Solomon, M. J., et al. (1992), Mol. Biol. Cell, 3:13-27). In addition, enzyme activity is negatively regulated by phosphorylations at Tyr-15 and/or Thr-14 (see, e.g., Solomon, M. J., et al., supra; Gu, Y., et al. (1992), EMBO J., 11:3995-4005; Krek, W., et al. (1991), EMBO J., 10:3331-3341; Norbury, C., et al. (1991), EMBO J., 10:3321-3329; Parker, L. L., et al. (1992), Proc. Nat""l. Acad. Sci. U.S.A., 89:2917-2921; McGowan, C. H., et al. (1993), EMBO J., 12:75-85), or by complex formation with inhibitor proteins like p16 (see, Serrano, M., et al. (1993), Nature, 366:704-707; Kamb, A., et al. (1994), Nature, 264:436-440; Nobori, T., et al. (1994), Nature, 368:753-756), p27 (see, Polyak, K., et al. (1994), Cell, 78:59-66; Toyoshima, H., et al. (1994), Cell, 78:67-74), p28 (see, Hengst, L., et al. (1994), Proc. Nat""l. Acad. Sci. U.S.A., 91:5291-5295) and p21 (see, Gu, Y., et al. (1993), Nature, 366:707-710; Xiiong, Y., et al. (1993), Nature, 366:701-704; Harper, J. W., et al. (1993), Cell, 75:805-816; Dulic, V., et al. (1994), Cell, 76:1013-1023), the latter being inducible by p53. Especially noteworthy is the fact that deletions of the p16 gene are found in over 50% of all human malignant cell lines tested (see, Kamb, A., supra, Nobori, T., et al., supra), although much less so in primary tumor cells (see, Spruck III, C. H., et al. (1994), Nature, 370:183-184), implicating p16 functions as tumor suppressor protein. Thus, both the cell growth signals transmitted through many oncogene products and the growth inhibitory signals from several tumor suppressor proteins modulate the activity of CDKs. Although mutations in CDKs themselves have not been associated with cancer, cyclin overexpression has been linked to tumorigenesis (see, Hunter, T., et al. (1991), Cell, 66:1071-1074; Keyomarsi, K., et al. (1993), Proc. Nat""l. Acad. Sci. U.S.A., 90:1112-1116; Wang, T. C., et al. (1994), Nature, 369:669-671.) Hence, CDKs are a promising target for developing inhibitors with antineoplastic effects and for the treatment of cell-proliferative diseases.
The purine ring system is a key structural element of the substrates and ligands of many biosynthetic, regulatory and signal transduction proteins including cellular kinases, G proteins and polymerases. As such, the purine ring system has been a good starting point in the search for inhibitors of many biomedically significant processes. In fact, while screening purine analogs for inhibition of various protein kinases, a relatively selective inhibitor, olomoucine (FIG. 1), was identified that competitively inhibited CDK2/cyclin A with an IC50 of 7 xcexcM (see, Vesely, J., et al., (1994) Eur. J. Biochem., 224:771-786). Further studies with olomoucine have demonstrated the orientation of the purine ring within the ATP-binding site of CDK2 is rotated almost 160 degrees relative to that of the adenosine ring of ATP. Consequently, it seems the introduction of new substituents at the 2, 6, and 9 positions of the purine ring rather than substituents appended to the ribose, as is normally done, might also selectively bind CDKs. There exists a need to rapidly screen compounds such as the trisubstituted purines to determine kinase inhibition. Quite surprisingly, the present invention satisfies such a need.
The present invention provides for methods of identifying compounds which modulate cell proliferation. The methods comprise the steps of (i) treating at least one cell with at least one compound, (ii) isolating a plurality of mRNA transcripts from said cell, and (iii) comparing a plurality of mRNA transcripts from a cell not treated with the compound to the mRNA transcripts from the treated cell, whereby a de in the number of mRNA transcripts indicates an inhibition of cell proliferation. In one embodiment of the invention, the compounds are inhibitors of cyclin-dependent kinases. In another embodiment, the mRNA transcripts are converted to cRNA. In yet another embodiment, the mRNA transcripts encode proteins associated with cell proliferation. Finally, in another embodiment, the mRNA is isolated by hybridization under stringent conditions to oligonucleotide probes of about 15 to about 50 nucleotides complementary to nucleic acids which encode proteins associated with cell proliferation. In a particularly preferred embodiment, the oligonucleotides are linked to a solid support in a high density array.
In another aspect of the invention, a method of determining the identity of proteins that modulate cell proliferation during or posure to chemical or genetic challenges is provided. The method comprises the steps of (i) isolating mRNA transcripts generated from cells after exposure to compounds known to modulate cellular proliferation, (ii) isolating mRNA transcripts generated from cells not exposed to said compounds, (iii) comparing the total number of mRNA transcripts from both treated and untreated cells, and (iv) determining which proteins are encoded by mRNA transcripts present in differing amounts in treated or untreated cells. In one embodiment of this aspect, the compounds are cyclin-dependent kinase inhibitors. In another embodiment, the mRNA transcripts are converted to cRNA. In still another embodiment, the mRNA is isolated by hybridization under stringent conditions to oligonucleotides of about 15 to about 50 nucleotides in length which are complementary to nucleic acids that encode proteins associated with cell proliferation. In a particularly preferred embodiment, the oligonucleotides are linked to a solid supporte in a high density array.
In a final aspect of this invention, a method of determining proteins associated with increased drug resistance is provided. The method comprises the steps of (i) isolating mRNA transcripts generated from drug-resistant cells after exposure to drugs known to inhibit cellular proliferation, (ii) isolating mRNA transcripts generated from non-drug resistant cells exposed to said drugs, (iii) comparing the total number of mRNA transcripts from both drug-resistant and non-resistant cells, and (iv) determining which proteins are encoded by mRNA transcripts present in increased amounts in the drug-resistant cells. In one embodiment, the compounds are cyclin-dependent kinase inhibitors. In another embodiment, the mRNA transcripts are converted to cRNA. In still another embodiment, the mRNA is isolated by hybridization under stringent conditions to oligonucleotides of about 15 to about 50 nucleotides in length which are complementary to nucleic acids that encode proteins associated with cell proliferation. In a particularly preferred embodiment, the oligonucleotides are linked to a solid supporte in a high density array.
Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description which follows.